DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

One-pot synthesis of heterodimeric agonists that activate the canonical Wnt signaling pathway.

Fragment antigen-binding domains (Fabs) from anti-Frizzled and anti-LRP6 monoclonal antibodies were conjugated using SpyTag-SpyCatcher chemistry via a one-pot reaction. The resulting synthetic heterodimeric agonist outperformed the natural ligand, Wnt-3a, in activating canonical Wnt signaling in mammalian cells. This approach should be broadly applicable to activate receptor-mediated cellular signaling.

Both positional and chemical variables control in vitro proteolytic cleavage of a presenilin ortholog.

Mechanistic details of intramembrane aspartyl protease (IAP) chemistry, which is central to many biological and pathogenic processes, remain largely obscure. Here, we investigated the in vitro kinetics of a microbial intramembrane aspartyl protease (mIAP) fortuitously acting on the renin substrate angiotensinogen and the C-terminal transmembrane segment of amyloid precursor protein (C100), which is cleaved by the presenilin subunit of γ-secretase, an Alzheimer disease (AD)-associated IAP. mIAP variants with substitutions in active-site and putative substrate-gating residues generally exhibit impaired, but not abolished, activity toward angiotensinogen and retain the predominant cleavage site (His-Thr). The aromatic ring, but not the hydroxyl substituent, within Tyr of the catalytic Tyr-Asp (YD) motif plays a catalytic role, and the hydrolysis reaction incorporates bulk water as in soluble aspartyl proteases. mIAP hydrolyzes the transmembrane region of C100 at two major presenilin cleavage sites, one corresponding to the AD-associated Aß42 peptide (Ala-Thr) and the other to the non-pathogenic Aß48 (Thr-Leu). For the former site, we observed more favorable kinetics in lipid bilayer-mimicking bicelles than in detergent solution, indicating that substrate-lipid and substrate-enzyme interactions both contribute to catalytic rates. High-resolution MS analyses across four substrates support a preference for threonine at the scissile bond. However, results from threonine-scanning mutagenesis of angiotensinogen demonstrate a competing positional preference for cleavage. Our results indicate that IAP cleavage is controlled by both positional and chemical factors, opening up new avenues for selective IAP inhibition for therapeutic interventions.

Microparticle-mediated sequestration of cell-secreted proteins to modulate chondrocytic differentiation.

Protein delivery is often used in tissue engineering applications to control differentiation processes, but is limited by protein instability and cost. An alternative approach is to control the cellular microenvironment through biomaterial-mediated sequestration of cell-secreted proteins important to differentiation. Thus, we utilized heparin-based microparticles to modulate cellular differentiation via protein sequestration in an in vitro model system of endochondral ossification. Heparin and poly(ethylene-glycol) (PEG; a low-binding material control)-based microparticles were incorporated into ATDC5 cell spheroids or incubated with ATDC5 cells in transwell culture. Reduced differentiation was observed in the heparin microparticle group as compared to PEG and no microparticle-containing groups. To determine if observed changes were due to sequestration of cell-secreted protein, the proteins sequestered by heparin microparticles were analyzed using SDS-PAGE and mass spectrometry. It was found that heparin microparticles bound insulin-like growth factor binding proteins (IGFBP)-3 and 5. When incubated with a small-molecule inhibitor of IGFBPs, NBI 31772, a similar delay in differentiation of ATDC5 cells was observed. These results indicate that heparin microparticles modulated chondrocytic differentiation in this system via sequestration of cell-secreted protein, a technique that could be beneficial in the future as a means to control cellular differentiation processes. STATEMENT OF SIGNIFICANCE: In this work, we present a proof-of-principle set of experiments in which heparin-based microparticles are shown to modulate cellular differentiation through binding of cell-secreted protein. Unlike existing systems that rely on expensive protein with limited half-lives to elicit changes in cellular behavior, this technique focuses on temporal modulation of cell-generated proteins. This technique also provides a biomaterials-based method that can be used to further identify sequestered proteins of interest. Thus, this work indicates that glycosaminoglycan-based biomaterial approaches could be used as substitutes or additions to traditional methods for modulating and identifying the cell-secreted proteins involved in directing cellular behavior.

Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete.

Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase.

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation.

Post-translational modifications of proteins regulate diverse cellular functions, with mounting evidence suggesting that hierarchical cross-talk between distinct modifications may fine-tune cellular responses. For example, in apoptosis, caspases promote cell death via cleavage of key structural and enzymatic proteins that in some instances is inhibited by phosphorylation near the scissile bond. In this study, we systematically investigated how protein phosphorylation affects susceptibility to caspase cleavage using an N-terminomic strategy, namely, a modified terminal amino isotopic labeling of substrates (TAILS) workflow, to identify proteins for which caspase-catalyzed cleavage is modulated by phosphatase treatment. We validated the effects of phosphorylation on three of the identified proteins and found that Yap1 and Golgin-160 exhibit decreased cleavage when phosphorylated, whereas cleavage of MST3 was promoted by phosphorylation. Furthermore, using synthetic peptides we systematically examined the influence of phosphoserine throughout the entirety of caspase-3, -7, and -8 recognition motifs and observed a general inhibitory effect of phosphorylation even at residues considered outside the classical consensus motif. Overall, our work demonstrates a role for phosphorylation in controlling caspase-mediated cleavage and shows that N-terminomic strategies can be tailored to study cross-talk between phosphorylation and proteolysis.

Protons as second messenger regulators of G protein signaling.

In response to environmental stress, cells often generate pH signals that serve to protect vital cellular components and reprogram gene expression for survival. A major barrier to our understanding of this process has been the identification of signaling proteins that detect changes in intracellular pH. To identify candidate pH sensors, we developed a computer algorithm that searches proteins for networks of proton-binding sidechains. This analysis indicates that Gα subunits, the principal transducers of G protein-coupled receptor (GPCR) signals, are pH sensors. Our structure-based calculations and biophysical investigations reveal that Gα subunits contain networks of pH-sensing sidechains buried between their Ras and helical domains. Further, we show that proton binding induces changes in conformation that promote Gα phosphorylation and suppress receptor-initiated signaling. Together, our computational, biophysical, and cellular analyses reveal an unexpected function for G proteins as mediators of stress-response signaling.

Application of multiplexed kinase inhibitor beads to study kinome adaptations in drug-resistant leukemia.

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCß, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.

Proteomic analysis of arsenic-exposed zebrafish (Danio rerio) identifies altered expression in proteins involved in fibrosis and lipid uptake in a gender-specific manner.

The zebrafish (Danio rerio) was used to investigate protein expression in the liver following arsenic exposure. Several disorders have been linked to arsenic exposure, including cancer, diabetes, and cardiovascular disease. The mechanisms of arsenic toxicity are poorly understood. Prior studies have described altered gene expression, inflammation, and mitogenic signaling in acute or chronic exposure models. A proteomic approach was employed to investigate arsenic-induced alteration in the zebrafish liver proteome following a 7-day exposure to 50 ppb sodium arsenite. Over 740 unique proteins were identified, with fewer than 2% showing differential expression. Molecular pathway analysis software identified lipid metabolism and transport as potential molecular targets. Immunoblots were used to confirm protein expression changes, whereas qPCR was employed to investigate gene expression changes. Overall, 25 proteins were differentially expressed in a gender-specific manner, 11 in males and 14 in females. Of these 25, a single protein, hydroxysteroid dehydrogenase like 2, showed decreased expression in both males and females following arsenic exposure. These findings indicate that protein expression is altered following arsenic exposure. The changes presented here seem to be most prevalent in lipid transport and metabolic pathways, suggesting a potential increase in fibrosis in males and decreased lipid accumulation and uptake in females.

Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.

Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple-negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, and prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 inhibition, but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

Hemorrhagic activity of HF3, a snake venom metalloproteinase: insights from the proteomic analysis of mouse skin and blood plasma.

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and blood extravasation. The mechanism of action of SVMPs has been investigated using various methodologies however the precise molecular events associated with microvessel disruption remains not fully understood. To gain insight into the hemorrhagic process, we analyzed the global effects of HF3, an extremely hemorrhagic SVMP from Bothrops jararaca, in the mouse skin and plasma. We report that in the HF3-treated skin there was evidence of degradation of extracellular matrix (collagens and proteoglycans), cytosolic, cytoskeleton, and plasma proteins. Furthermore, the data suggest that direct and indirect effects promoted by HF3 contributed to tissue injury as the activation of collagenases was detected in the HF3-treated skin. In the plasma analysis after depletion of the 20 most abundant proteins, fibronectin appeared as degraded by HF3. In contrast, some plasma proteinase inhibitors showed higher abundance compared to control skin and plasma. This is the first study to assess the complex in vivo effects of HF3 using high-throughput proteomic approaches, and the results underscore a scenario characterized by the interplay between the hydrolysis of intracellular, extracellular, and plasma proteins and the increase of plasma inhibitors in the hemorrhagic process.

The plasma microparticle proteome.

All cell types shed ectosomes and exosomes, collectively known as microparticles (MP; 0.1 to 1.5 µm in diameter), when activated or stressed; normal human plasma contains ~2 µg MP protein/mL. The cellular composition of plasma MP is altered in many diseases, including acute coronary syndrome, diabetes mellitus, sepsis, and sickle cell disease. We measured the plasma MP protein composition of 42 patients (median age 69.5 years, most with cardiovascular disease) by label-free liquid chromatography coupled to tandem mass spectrometry. Among 458 proteins detected with high confidence (identified by at least two unique peptides with SEQUEST XCor (Thermo Electron Corp., San Jose, CA) ≥ 2.0, 2.2, and 3.3 for charge states +1, +2, and +3, respectively), 130 were present in most patients, representing a "core" set of plasma MP proteins. This core is enriched in cytoskeletal, integrin complex, and hemostasis proteins, and spectral counts of several proteins correlate with patient age and gender. We conclude that the MP proteome may be a useful and reliable source of biologically relevant disease biomarkers.

Plasma-derived microparticles for biomarker discovery.

The field of mass spectrometry-based proteomics has been transformed over the last decade due to advances in technology, sample preparation, bioinformatics, and computational tools. While this has led to a dramatic increase in research related to biomarker discovery, the promise of finding a significant number of new biomarkers has not yet materialized. Current proteomic technology is able to detect and analyze extremely small amounts of proteins (picomole to attomole level), but has difficulty detecting and quantifying proteins present at 2- to 3-orders of magnitude lower than the more abundant proteins. This is referred to as the dynamic range problem. Normal biological fluids used for biomarker discovery, such as plasma or urine, contain a small number of proteins present at much higher amounts than the remaining proteins. For example, in the plasma, albumin and immunoglobulins are present at milligrams per milliliter, while proteins of interest for biomarker discovery may be present at micrograms to picograms per ml. This has led us to investigate the microparticle subproteome which has a high likelihood of containing potential biomarkers. While this subproteome makes up less than 0.01% of the total plasma proteome, it is rich in proteins altered under a variety of pathological conditions.

Isolation and identification of potential urinary microparticle biomarkers of bladder cancer.

Bladder cancer leads to approximately 13,000 deaths annually in the United States. Early disease is often treated with minimal morbidity and has good prognosis, while the opposite is true for advanced disease. Currently, no tools exist for early detection of this cancer. Microparticles are small, subcellular particles released by essentially all cells upon activation and are known to be produced constitutively by cancer cells. Since most bladder cancers originate in the urothelial cells lining the lumen of the organ, we hypothesize that these cells will release microparticles into the urine. The goal of this study was to identify potential biomarkers in the urinary microparticles of individuals with bladder cancer. Urine microparticles from five healthy individuals and four individuals with bladder cancer were isolated. Samples were delipidated by PAGE and trypsin-digested, peptides were extracted, and the proteome was examined by LC-MS/MS using a Thermo Finnigan LTQ and LTQ-FT ion trap mass spectrometer. Protein identification was determined by SEQUEST and relative quantitation was assessed by comparing spectral counts. Eight proteins were elevated in the microparticles from individuals with bladder cancer. They include five proteins associated with the epidermal growth factor receptor pathway, the alpha subunit of GsGTP binding protein, resistin, and retinoic acid-induced protein 3. Further studies will be needed to validate these potential biomarkers.

Statistical identification of differentially labeled peptides from liquid chromatography tandem mass spectrometry.

LC-MS/MS with certain labeling techniques such as isotope-coded affinity tag (ICAT) enables quantitative analysis of paired protein samples. However, current identification and quantification of differentially expressed peptides (and proteins) are not reliable for large proteomics screening of complex biological samples. The number of replicates is often limited because of the high cost of experiments and the limited supply of samples. Traditionally, a simple fold change cutoff is used, which results in a high rate of false positives. Standard statistical methods such as the two-sample t-test are unreliable and severely underpowered due to high variability in LC-MS/MS data, especially when only a small number of replicates are available. Using an advanced error pooling technique, we propose a novel statistical method that can reliably identify differentially expressed proteins while maintaining a high sensitivity, particularly with a small number of replicates. The proposed method was applied both to an extensive simulation study and a proteomics comparison between microparticles (MPs) generated from platelet (platelet MPs) and MPs isolated from plasma (plasma MPs). In these studies, we show a significant improvement of our statistical analysis in the identification of proteins that are differentially expressed but not detected by other statistical methods. In particular, several important proteins - two peptides for beta-globin and three peptides for von Willebrand Factor (vWF) - were identified with very small false discovery rates (FDRs) by our method, while none was significant when other conventional methods were used. These proteins have been reported with their important roles in microparticles in human blood cells: vWF is a platelet and endothelial cell product that binds to P-selectin, GP1b, and GP IIb/IIIa, and beta-globin is one of the peptides of hemoglobin involved in the transportation of oxygen by red blood cells.

Proteomic discovery of 21 proteins expressed in human plasma-derived but not platelet-derived microparticles.

Microparticles (MPs) are small membrane vesicles generated by essentially all cell types. In the plasma, most MPs are derived from platelets, but those from other sources, particularly leukocytes (macrophages, lymphocytes, and neutrophils), endothelial cells, and even smooth muscle cells can be detected and appear to play an important role in normal physiology and various diseases. In previous work we analyzed the proteome of MPs generated from isolated platelets (platelet MPs). Here, we report on a comparative analysis of microparticles isolated from plasma (plasma MPs) versus platelet MP using two complementary methods of comparative analysis. The first method, spectral count analysis, yielded 21 proteins detected in plasma MPs (with a total spectral count of 10 or greater) that were essentially absent in platelet MPs (with a total spectral count of 1 or 0). An additional two proteins (von Willebrand Factor, albumin) were present in both types of MPs but enriched in the plasma MPs. The second method, isotope-coded affinity tag (ICAT) labeling of proteins, supported the spectral count results for the more abundant proteins and provided better relative quantitation of differentially expressed proteins. Proteins present only in the plasma MPs include several associated with apoptosis (CD5-like antigen, galectin 3 binding protein, several complement components), iron transport (transferrin, transferrin receptor, haptoglobin), immune response (complement components, immunoglobulin J and kappa chains), and the coagulation process (protein S, coagulation factor VIII).

The platelet microparticle proteome.

Platelet-derived microparticles are the most abundant type of microparticle in human blood and contribute to many biologically significant processes. Here, we report the first proteomic analysis of microparticles generated from activated platelets. Using 1D SDS-PAGE and liquid chromatography coupled to a linear ion trap mass spectrometer, the identification of 578 proteins was accomplished using a minimum of 5 MS/MS detections of at least two different peptides for each protein. These microparticles displayed many proteins intrinsic to and well-characterized on platelets. For example, microparticles in these experiments were found to contain membrane surface proteins including GPIIIa, GPIIb, and P-selectin, as well other platelet proteins such as the chemokines CXCL4, CXCL7, and CCL5. In addition, approximately 380 of the proteins identified were not found in two previous studies of the platelet proteome. Since several of the proteins detected here have been previously implicated in microparticle formation and/or pathological function, it is hoped that this study will help fuel future work concerning the possible role of microparticles in various disease states.

In vivo visualization of reactive oxidants and leukocyte-endothelial adherence following hemorrhagic shock.

The generation of oxygen radicals during leukocyte-endothelial cell interaction is considered to represent one of the fundamental steps of microvascular injury following ischemia and reperfusion. Indirect evidence also suggests that this relationship may be important following hemorrhagic shock. The purpose of this study was to characterize the temporal changes of reactive oxygen species (ROS) in the mesenteric microvascular endothelium, in vivo, as a consequence of hemorrhagic shock and reperfusion, and to correlate this ROS production to leukocyte adherence. Following a control period, blood was withdrawn to reduce the mean arterial pressure to 40 mmHg for 1 h in urethane-anesthetized rats. Mesenteric venules in a transilluminated segment of small intestine were examined to quantitate changes in ROS generation and leukocyte adherence. Sprague-Dawley rats were injected with dihydrorhodamine 123, a hydroperoxide-sensitive fluorescent probe that is trapped within viable cells as a nonfluorescent form and then converted to the mitochondrion-selective form rhodamine 123 by hydroperoxides. The fluorescent light emission from rhodamine 123 was recorded with digital microscopy and downloaded to a computerized image analysis program. Our results demonstrated an 80% increase in ROS generation beginning within 5 min into resuscitation and a 10-fold increase in leukocyte adherence that occurred at 10 min after resuscitation. Both ROS generation and leukocyte adherence were attenuated with pre-shock administration of platelet activating factor (PAF) antagonist, WEB 2086, and the CD11/CD18a antibody, anti-LFA-1beta. Our findings suggest that ROS production in endothelial cells is increased during reperfusion following hemorrhagic shock and that the mechanism of expression is mediated in part by both PAF expression and subsequent leukocyte adherence.

Native low-density lipoprotein induces endothelial nitric oxide synthase dysfunction: role of heat shock protein 90 and caveolin-1.

Although native LDL (n-LDL) is well recognized for inducing endothelial cell (EC) dysfunction, the mechanisms remain unclear. One hypothesis is n-LDL increases caveolin-1 (Cav-1), which decreases nitric oxide (*NO) production by binding endothelial nitric oxide synthase (eNOS) in an inactive state. Another is n-LDL increases superoxide anion (O(2)(*-)), which inactivates *NO. To test these hypotheses, EC were incubated with n-LDL and then analyzed for *NO, O(2)(*-), phospho-eNOS (S1179), eNOS, Cav-1, calmodulin (CaM), and heat shock protein 90 (hsp90). n-LDL increased NOx by more than 4-fold while having little effect on A23187-stimulated nitrite production. In contrast, n-LDL decreased cGMP under basal and A23187-stimulated conditions and increased O(2)(*-) by a mechanism that could be inhibited by L-nitroargininemethylester (L-NAME) and BAPTA/AM. n-LDL increased phospho-eNOS by 149%, eNOS by approximately 34%, and Cav-1 by 28%, and decreased the association of hsp90 with eNOS by 49%. n-LDL did not appear to alter eNOS distribution between membrane fractions (approximately 85%) and cytosol (approximately 15%). Only 3-6% of eNOS in membrane fractions was associated with Cav-1. These data support the hypothesis that n-LDL increases O(2)(*-), which scavenges *NO, and suggest that n-LDL uncouples eNOS activity by decreasing the association of hsp90 as an initial step in signaling eNOS to generate O(2)(*-).